US6040439A - Method for chemical synthesis of oligonucleotides - Google Patents
Method for chemical synthesis of oligonucleotides Download PDFInfo
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- US6040439A US6040439A US09/145,973 US14597398A US6040439A US 6040439 A US6040439 A US 6040439A US 14597398 A US14597398 A US 14597398A US 6040439 A US6040439 A US 6040439A
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- phosphoroamidite
- unprotected
- oligonucleotide
- base moiety
- nucleoside
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
Definitions
- the present invention relates to a method for chemical synthesis of oligonucleotides.
- the present invention relates to a novel method capable of chemically synthesizing a long-chain DNA or RNA fragment easily and reliably from a base moiety-unprotected nucleotide phosphoroamidite as a unit, as well as to a novel compound used in said method.
- the phosphoroamidite method is used most widely at present as a method of chemically synthesizing oligonucleotides such as DNA fragments and RNA fragments (Nucleic Acid Research, 17:7059-7071, 1989).
- this phosphoroamidite method makes use of a condensation reaction between a nucleoside phosphoroamidite and a nucleoside as a key reaction using tetrazole as an accelerator. Because this reaction usually occurs competitively on both the hydroxyl group in a sugar moiety and the amino group in a nucleoside base moiety, the selective reaction on only the hydroxyl group in a sugar moiety is required to synthesize a desired nucleotide. Accordingly, the side reaction on the amino group was prevented in the prior art by protecting the amino group, as illustrated in the following reaction scheme: ##STR2##
- condensation yield in each step is low (about 97%: at least 99% yield is required for synthesis of a 50-mer or more long-chain oligonucleotide) and a commercial automatic DNA synthesizer cannot be used for this method, so a long-chain oligonucleotide consisting of 50 to 100 nucleotides generally required in chemical synthesis of DNA etc. cannot be synthesized;
- pyridine hydrochloride used as an accelerator is an unstable compound with very high moistureproofness, and thus its handling is difficult.
- the present invention was made in view of the prior art described above, and the object of the present invention is to provide a practical method capable of chemically synthesizing a 100-mer or more long-chain oligonucleotide easily and reliably as well as a novel compound used in said method.
- the present invention provides a method for chemical synthesis of an oligonucleotide by the phosphoroamidite method, which comprises preparing a base moiety-unprotected nucleoside phosphoroamidite from a base moiety-unprotected nucleoside by use of an imidazole trifluoromethanesulfonate represented by the following chemical formula, and coupling said base moiety-unprotected nucleotide phosphoroamidite in a predetermined order to chemically synthesize an oligonucleotide consisting of a specific nucleotide sequence. ##STR4##
- the coupled, base moiety-unprotected nucleoside phosphoroamidite is treated with a benzimidazole trifluoromethanesulfonate solution.
- this invention also provides an imidazole trifluoromethanesulfonate represented by the chemical formula. ##STR5##
- the present inventors found that a base moiety-unprotected nucleoside phosphoroamidite prepared by use of a novel compound, imidazole trifluoromethanesulfonate (referred to hereinafter as imidazolium triflate) in place of the conventionally used tetrazole as an accelerator for condensation reaction between nucleoside phosphoroamidite and nucleotide is free of the side reaction on the amino group in the nucleotide base moiety thereof, and as a result, they found that complicated procedures such as, for example, introduction and removal of a protective group are not required, and also that its synthesis can be conducted by a commercial synthesizer, thereby completing this invention.
- imidazole trifluoromethanesulfonate referred to hereinafter as imidazolium triflate
- the present inventors found that the side reaction on the amino group in the base moiety can be completely inhibited by treating the above-described coupled, base moiety-unprotected nucleoside phosphoroamidite with a methanol solution of a benzimidazole trifluoromethanesulfonate (referred to hereinafter as benzimidazolium triflate) whereby a more perfect oligonucleotide is synthesized, and the present invention was thereby completed.
- benzimidazolium triflate a benzimidazole trifluoromethanesulfonate
- FIG. 1 is a schematic drawing of each reaction step in the method of this invention.
- FIG. 2 is a schematic drawing of each reaction step in the method of the present invention where ammonia treatment was performed.
- FIG. 3 is a HPLC profile of DNA fragments synthesized in the method of this invention.
- the imidazolium triflate of the present invention can be prepared by mixing imidazole with trifluoromethanesulfonic acid in 1:1 equivalents in dichloromethane, as illustrated below in its preparation example in Example 1.
- the imidazolium triflate thus obtained does not absorb moisture as also shown in Example 1 and is extremely stable under usual conditions for use, so it can be easily handled.
- a base moiety-unprotected nucleoside phosphoroamidite is prepared from a base moiety-unprotected nucleotide by use of the imidazolium triflate as described above, and this base moiety-unprotected nucleoside phosphoroamidite is used as a unit and each nucleoside phosphoroamidite is coupled in a predetermined order thereby chemically synthesizing an oligonucleotide consisting of a specific nucleotide sequence.
- the base moiety-unprotected nucleoside phosphoroamidite can be prepared by reacting the base moiety-unprotected nucleoside phosphoroamidite with cyanoethyl-bis-amidite in the presence of the imidazolium triflate as a catalyst as illustrated e.g. in Example 2 below.
- the reaction occurs selectively on the hydroxide group in the sugar moiety of the nucleoside, so four kinds of N-unprotected nucleoside phosphoroamidites used in DNA synthesis, that is, deoxyadenosine, deoxythymidine, deoxyguanosine and thymidine phosphoroamidites can be obtained quantitatively.
- the four kinds of N-unprotected nucleoside phosphoroamidites thus obtained are used as units to synthesize an oligonucleotide consisting of a desired nucleotide sequence by the solid-phase synthetic method etc. known in the art. Further, this synthetic reaction can also be conducted in a commercial DNA synthesizer by a method according to its protocol.
- each coupled N-unprotected nucleoside phosphoroamidite is preferably subjected after each coupling to treatment with a solution (e.g. an ethanol solution) of benzimidazolium triflate.
- a solution e.g. an ethanol solution
- benzimidazolium triflate e.g. benzimidazolium triflate
- the benzimidazolium triflate can be synthesized in the following reaction scheme: ##STR6##
- Example 1 The imidazolium triflate obtained in Example 1 was used as the catalyst so that a base moiety-unprotected nucleoside was reacted with cyanoethyl-bis-amidite, as shown in the following reaction scheme: ##STR8##
- the four kinds of N-unprotected nucleoside phosphoroamidites shown in Table 2, that is, deoxyadenosine, deoxythymidine, deoxyguanosine and thymidine phosphoroamidites were prepared respectively.
- the respective nucleoside phosphoroamidites were obtained almost quantitatively.
- Example 3 From the 4 kinds of N-unprotected nucleoside phosphoroamidites as units obtained in Example 3 [sic.], a 60-mer DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1 was synthesized by the solid-phase synthetic method using a commercial DNA synthesizer. The reaction cycle was as shown in Table 3.
- each step (condensation reaction) in the chain-elongation shown in Table 1 proceeded in almost 100% yield, and a phosphate moiety-protected 60-mer oligonucleotide was obtained usually in 100% yield.
- This yield was extremely high in considering that the yield of a 60-mer oligonucleotide by generally conducted conventional methods is about 20 to 40%.
- the imidazolium triflate that is the novel compound of this invention and the method of synthesizing oligonucleotides by use of this imidazolium triflate have the following advantages:
- condensation yield in each step is as high as 100%, and the present method can also be applied to an automatic synthesizer by merely changing a program for synthesis and reagents used, so synthesis of a long-chain oligonucleotide consisting of 50 to 100 nucleotides generally required in chemical synthesis of DNA etc. is feasible in 1/10 or less costs as compared with those of conventional methods;
- the imidazolium triflate of this invention used as an accelerator is a stable compound which does not absorb moisture, so its handling under usually conditions for use is very easy.
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Abstract
The present invention provides a practical method capable of chemically synthesizing a 100-mer or more long-chain oligonucleotide easily and reliably and a novel compound used in said method. The present invention relates to a method for chemical synthesis of an oligonucleotide by the phosphoroamidite method, which comprises preparing a base moiety-unprotected nucleoside phosphoroamidite from a base moiety-unprotected nucleoside by use of an imidazole trifluoromethanesulfonate represented by the following chemical formula, and coupling said base moiety-unprotected nucleotide phosphoroamidite in a predetermined order to chemically synthesize an oligonucleotide consisting of a specific nucleotide sequence, as well as to an imidazole trifluoromethanesulfonate represented by the chemical formula. ##STR1##
Description
1. Field of the Invention
The present invention relates to a method for chemical synthesis of oligonucleotides. In particular, the present invention relates to a novel method capable of chemically synthesizing a long-chain DNA or RNA fragment easily and reliably from a base moiety-unprotected nucleotide phosphoroamidite as a unit, as well as to a novel compound used in said method.
2. Description of the Related Art
The phosphoroamidite method is used most widely at present as a method of chemically synthesizing oligonucleotides such as DNA fragments and RNA fragments (Nucleic Acid Research, 17:7059-7071, 1989). In general, this phosphoroamidite method makes use of a condensation reaction between a nucleoside phosphoroamidite and a nucleoside as a key reaction using tetrazole as an accelerator. Because this reaction usually occurs competitively on both the hydroxyl group in a sugar moiety and the amino group in a nucleoside base moiety, the selective reaction on only the hydroxyl group in a sugar moiety is required to synthesize a desired nucleotide. Accordingly, the side reaction on the amino group was prevented in the prior art by protecting the amino group, as illustrated in the following reaction scheme: ##STR2##
However, the protective group should be removed when synthesis was finished, and operationally complicated organic reactions and a large amount of expensive and harmful reagents are required to introduce and remove said protective group, which in view of practical usability, economical efficiency, environmental protection etc., is a great problem in carrying out this prior method. Accordingly, there is demand for a method of chemically synthesizing an oligonucleotide from an amino group-unprotected nucleoside phosphoroamidite as a unit, and the method of Letsinger et al., as shown in the following reaction scheme, is known as a pioneering method (Nucleic Acids Research, 20:1879-1882, 1992): ##STR3##
However, the method of Letsinger et al. is not practical, not universal and is not used in practice since there are following disadvantages:
(1) condensation yield in each step is low (about 97%: at least 99% yield is required for synthesis of a 50-mer or more long-chain oligonucleotide) and a commercial automatic DNA synthesizer cannot be used for this method, so a long-chain oligonucleotide consisting of 50 to 100 nucleotides generally required in chemical synthesis of DNA etc. cannot be synthesized;
(2) highly reactive, specific nucleoside phosphoroamidites only can be used, and thus this method has a limited scope of application and is not practical; and
(3) pyridine hydrochloride used as an accelerator is an unstable compound with very high moistureproofness, and thus its handling is difficult.
The present invention was made in view of the prior art described above, and the object of the present invention is to provide a practical method capable of chemically synthesizing a 100-mer or more long-chain oligonucleotide easily and reliably as well as a novel compound used in said method.
To solve the problem, the present invention provides a method for chemical synthesis of an oligonucleotide by the phosphoroamidite method, which comprises preparing a base moiety-unprotected nucleoside phosphoroamidite from a base moiety-unprotected nucleoside by use of an imidazole trifluoromethanesulfonate represented by the following chemical formula, and coupling said base moiety-unprotected nucleotide phosphoroamidite in a predetermined order to chemically synthesize an oligonucleotide consisting of a specific nucleotide sequence. ##STR4##
In a preferable embodiment of the method of this invention, the coupled, base moiety-unprotected nucleoside phosphoroamidite is treated with a benzimidazole trifluoromethanesulfonate solution.
Further, this invention also provides an imidazole trifluoromethanesulfonate represented by the chemical formula. ##STR5##
That is, the present inventors found that a base moiety-unprotected nucleoside phosphoroamidite prepared by use of a novel compound, imidazole trifluoromethanesulfonate (referred to hereinafter as imidazolium triflate) in place of the conventionally used tetrazole as an accelerator for condensation reaction between nucleoside phosphoroamidite and nucleotide is free of the side reaction on the amino group in the nucleotide base moiety thereof, and as a result, they found that complicated procedures such as, for example, introduction and removal of a protective group are not required, and also that its synthesis can be conducted by a commercial synthesizer, thereby completing this invention. Further, the present inventors found that the side reaction on the amino group in the base moiety can be completely inhibited by treating the above-described coupled, base moiety-unprotected nucleoside phosphoroamidite with a methanol solution of a benzimidazole trifluoromethanesulfonate (referred to hereinafter as benzimidazolium triflate) whereby a more perfect oligonucleotide is synthesized, and the present invention was thereby completed.
FIG. 1 is a schematic drawing of each reaction step in the method of this invention.
FIG. 2 is a schematic drawing of each reaction step in the method of the present invention where ammonia treatment was performed.
FIG. 3 is a HPLC profile of DNA fragments synthesized in the method of this invention.
Hereinafter, the best mode for carrying out the present invention is described in detail.
The imidazolium triflate of the present invention can be prepared by mixing imidazole with trifluoromethanesulfonic acid in 1:1 equivalents in dichloromethane, as illustrated below in its preparation example in Example 1.
The imidazolium triflate thus obtained does not absorb moisture as also shown in Example 1 and is extremely stable under usual conditions for use, so it can be easily handled.
In the chemical synthetic method of this invention, a base moiety-unprotected nucleoside phosphoroamidite is prepared from a base moiety-unprotected nucleotide by use of the imidazolium triflate as described above, and this base moiety-unprotected nucleoside phosphoroamidite is used as a unit and each nucleoside phosphoroamidite is coupled in a predetermined order thereby chemically synthesizing an oligonucleotide consisting of a specific nucleotide sequence.
The base moiety-unprotected nucleoside phosphoroamidite can be prepared by reacting the base moiety-unprotected nucleoside phosphoroamidite with cyanoethyl-bis-amidite in the presence of the imidazolium triflate as a catalyst as illustrated e.g. in Example 2 below. In this case, the reaction occurs selectively on the hydroxide group in the sugar moiety of the nucleoside, so four kinds of N-unprotected nucleoside phosphoroamidites used in DNA synthesis, that is, deoxyadenosine, deoxythymidine, deoxyguanosine and thymidine phosphoroamidites can be obtained quantitatively.
The four kinds of N-unprotected nucleoside phosphoroamidites thus obtained are used as units to synthesize an oligonucleotide consisting of a desired nucleotide sequence by the solid-phase synthetic method etc. known in the art. Further, this synthetic reaction can also be conducted in a commercial DNA synthesizer by a method according to its protocol.
In the method of this invention, each coupled N-unprotected nucleoside phosphoroamidite is preferably subjected after each coupling to treatment with a solution (e.g. an ethanol solution) of benzimidazolium triflate. By this treatment, the side reaction on the amino group in the base moiety is completely inhibited, and a more perfect oligonucleotide is thus synthesized.
The benzimidazolium triflate can be synthesized in the following reaction scheme: ##STR6##
Hereinafter, the present invention is described in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention.
Preparation of imidazolium triflate
Imidazole and trifluoromethanesulfonic acid were mixed in 1:1 equivalents in dichloromethane and reacted at 25° C. for 10 minutes as shown in the reaction scheme below, whereby the imidazolium triflate of this invention was prepared. ##STR7##
As a result of analysis in conventional methods, the resulting imidazolium triflate had the characteristics shown in Table 1.
TABLE 1 ______________________________________ Colorless crystal Melting point: 197-198° C. Elementary analysis Theoretical: C.sub.4 H.sub.5 F.sub.3 N.sub.2 O.sub.3 S: C, 22.02; H, 2.31; N, 12.84 Found: C, 21.96; H, 2.30; N, 12.74 No moistureproofness ______________________________________
Preparation of base moiety-unprotected nucleoside phosphoroamidite
The imidazolium triflate obtained in Example 1 was used as the catalyst so that a base moiety-unprotected nucleoside was reacted with cyanoethyl-bis-amidite, as shown in the following reaction scheme: ##STR8##
By this reaction, the four kinds of N-unprotected nucleoside phosphoroamidites shown in Table 2, that is, deoxyadenosine, deoxythymidine, deoxyguanosine and thymidine phosphoroamidites were prepared respectively. As also shown in Table 2, the respective nucleoside phosphoroamidites were obtained almost quantitatively.
TABLE 2
__________________________________________________________________________
##STR9##
##STR10##
##STR11##
##STR12##
__________________________________________________________________________
yield, %:
96 98 97 99
purity, %:
>98 >98 >96 >99
.sup.31 P NMR, ppm:
149.0, 149.1
149.2, 149.3
149.1, 149.2 149.0, 149.1
__________________________________________________________________________
Synthesis of DNA fragment
From the 4 kinds of N-unprotected nucleoside phosphoroamidites as units obtained in Example 3 [sic.], a 60-mer DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 1 was synthesized by the solid-phase synthetic method using a commercial DNA synthesizer. The reaction cycle was as shown in Table 3.
TABLE 3 ______________________________________ Step Operation reagent(s) time, min ______________________________________ 1 washing CH.sub.3 CN 0.50 2ditritylation 3% CCl.sub.3 COOH/CH.sub.2 CH.sub.2 1.0 × 3 3 washing CH.sub.3 CN 2.0 4 coupling 0.1M amidite/CH.sub.3 CN + 0.25 0.1M IMT/CH.sub.3CN 5 wait 1.0 6 N-P cleavage 0.3M BIT/CH.sub.3 CN 0.50 7 wait 2.0 8 washing CH.sub.3 CN 0.50 9 oxidation 1M t-C.sub.4 H.sub.9 OOH/CH.sub.2 Cl.sub.2 0.25 10 wait 1.0 ______________________________________ BIT = benzimidazolium triflate; IMT = imidazolium triflate
In this synthetic reaction, each step (condensation reaction) in the chain-elongation shown in Table 1 proceeded in almost 100% yield, and a phosphate moiety-protected 60-mer oligonucleotide was obtained usually in 100% yield. This yield was extremely high in considering that the yield of a 60-mer oligonucleotide by generally conducted conventional methods is about 20 to 40%.
Further, as shown in FIG. 2, deprotection and elimination by treatment with an ammonia solution (25° C., 60 minutes) were carried out whereby the unprotected 60-mer DNA was obtained in quantitative yield.
Analysis of the resulting crude unprotected 60-mer DNA by high performance liquid chromatography under the conditions shown in Table 4 indicated that its purity was 95% or more as shown in FIG. 3.
TABLE 4
______________________________________
Analytical conditions
______________________________________
Column: DEAE-2.5μ (250 mm)
Flow rate: 0.5 mL/min
Temperature: 25° C.
Eluent:
A: 20 mM Tris-HCl (pH 9.0)
B: A + 1M NaCl
Gradient: A:B (100:0) → (50:50) linear
gradient
______________________________________
As described above in detail, the imidazolium triflate that is the novel compound of this invention and the method of synthesizing oligonucleotides by use of this imidazolium triflate have the following advantages:
(1) condensation yield in each step is as high as 100%, and the present method can also be applied to an automatic synthesizer by merely changing a program for synthesis and reagents used, so synthesis of a long-chain oligonucleotide consisting of 50 to 100 nucleotides generally required in chemical synthesis of DNA etc. is feasible in 1/10 or less costs as compared with those of conventional methods;
(2) because unspecified nucleotide phosphoroamidites can be used, the present method has a broad scope of application and is practical; and
(3) the imidazolium triflate of this invention used as an accelerator is a stable compound which does not absorb moisture, so its handling under usually conditions for use is very easy.
__________________________________________________________________________ # SEQUENCE LISTING - <160> NUMBER OF SEQ ID NOS: 1 - <210>SEQ ID NO 1 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: #Sequence: SYNTHETICION: Description of Artificial DNA - <400> SEQUENCE: 1 - tatgggcctt ttgataggat gctcaccgag caaaaccaag aacaaccagg ag -#attttatt 60 __________________________________________________________________________
Claims (2)
1. A method for chemically synthesizing an oligonucleotide by a phosphoroamidite method, which comprises:
reacting amino group-unprotected nucleosides individually with a phosphoroamidite reagent in the presence of imidazole trifluoromethanesulfonate represented by the following chemical formula: ##STR13## to prepare amino group-unprotected nucleoside phosphoroamidites; and coupling each of the prepared amino group-unprotected nucleoside phosphoroamidites in a predetermined order to chemically synthesize the oligonucleotide of a specific nucleotide sequence.
2. The method according to claim 1, wherein the coupled amino group-unprotected nucleoside phosphoroamidite is treated with a benzimidazole trifluoromethanesulfonate solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP241292 | 1997-09-05 | ||
| JP9241292A JPH1180185A (en) | 1997-09-05 | 1997-09-05 | Chemical synthesis of oligonucleotide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US6040439A true US6040439A (en) | 2000-03-21 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US09/145,973 Expired - Fee Related US6040439A (en) | 1997-09-05 | 1998-09-03 | Method for chemical synthesis of oligonucleotides |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US6040439A (en) |
| EP (1) | EP0906917B1 (en) |
| JP (1) | JPH1180185A (en) |
| CA (1) | CA2246909C (en) |
| DE (1) | DE69819998T2 (en) |
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| WO2015089053A1 (en) | 2013-12-09 | 2015-06-18 | Integrated Dna Technologies, Inc. | Long nucleic acid sequences containing variable regions |
| US9670517B1 (en) | 2012-01-16 | 2017-06-06 | Integrated Dna Technologies, Inc. | Synthesis of long nucleic acid sequences |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6274725B1 (en) | 1998-06-02 | 2001-08-14 | Isis Pharmaceuticals, Inc. | Activators for oligonucleotide synthesis |
| US6929907B2 (en) * | 1999-12-31 | 2005-08-16 | North Carolina State University | Methods and compositions for determining the purity of chemically synthesized nucleic acids |
| EP1721908A4 (en) | 2004-03-01 | 2009-12-16 | Japan Science & Tech Agency | Novel method of synthesizing nucleic acid without protecting nucleotide bases |
| AU2005315631A1 (en) * | 2004-12-15 | 2006-06-22 | Girindus Ag | Synthesis of phosphitylated compounds using a quaternary heterocyclic activator |
| RU2465280C2 (en) * | 2005-03-04 | 2012-10-27 | Гириндус Аг | Synthesis of oligonucleotides |
| IL185439A0 (en) * | 2005-03-04 | 2008-01-06 | Girindus Ag | Synthesis of oligonucleotides |
| JP2022177332A (en) * | 2019-10-24 | 2022-12-01 | 日東電工株式会社 | Method for producing oligonucleotide |
| CN114981281A (en) * | 2019-11-13 | 2022-08-30 | 日本新药株式会社 | Method for producing oligonucleotide compound |
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| WO1998029429A1 (en) * | 1996-12-27 | 1998-07-09 | Isis Pharmaceuticals, Inc. | Method for the synthesis of nucleotide or oligonucleotide phosphoramidites |
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1997
- 1997-09-05 JP JP9241292A patent/JPH1180185A/en active Pending
-
1998
- 1998-09-03 US US09/145,973 patent/US6040439A/en not_active Expired - Fee Related
- 1998-09-04 CA CA002246909A patent/CA2246909C/en not_active Expired - Fee Related
- 1998-09-04 DE DE69819998T patent/DE69819998T2/en not_active Expired - Fee Related
- 1998-09-04 EP EP98307128A patent/EP0906917B1/en not_active Expired - Lifetime
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| US5077285A (en) * | 1989-07-31 | 1991-12-31 | Merck & Co., Inc. | Imidazole compounds and their use as transglutaminase inhibitors |
| WO1998029429A1 (en) * | 1996-12-27 | 1998-07-09 | Isis Pharmaceuticals, Inc. | Method for the synthesis of nucleotide or oligonucleotide phosphoramidites |
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Also Published As
| Publication number | Publication date |
|---|---|
| EP0906917B1 (en) | 2003-11-26 |
| JPH1180185A (en) | 1999-03-26 |
| DE69819998D1 (en) | 2004-01-08 |
| CA2246909A1 (en) | 1999-03-05 |
| EP0906917A2 (en) | 1999-04-07 |
| EP0906917A3 (en) | 1999-09-22 |
| CA2246909C (en) | 2003-12-02 |
| DE69819998T2 (en) | 2004-09-02 |
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